The present invention relates generally to the discovery that selective alpha1B adrenergic receptor antagonists can be useful for the treatment of pain. In particular, the present invention relates to methods for screening compounds to identify an alpha1B adrenergic receptor ligand that binds to the alpha1B adrenergic receptor to provide an analgesic effect.
Alpha1 adrenergic receptors are G-protein coupled transmembrane receptors that mediate various actions of the sympathetic nervous system through the binding of the catecholamines, epinephrine and norepinephrine. Currently, several subtypes of the alpha1 adrenergic receptors are known to exist for which the genes have been cloned: alpha1A (previously known as alpha1C), alpha1B and alpha1D. The existence of an additional subtype, the alpha1L adrenergic receptor subtype, has been proposed; however, the gene for the alpha1L adrenergic receptor subtype has yet to be cloned. Although these subtypes can be pharmacologically distinguished, existing subtype-selective compounds are only moderately specific and may interact with more than one alpha1 adrenergic receptor subtype. (See Giardina, D., et al., J. Med. Chem., 1996, 39:4602-4607). Accordingly, therapeutic use of nonselective alpha1 adrenergic receptor antagonists must be carefully monitored as such antagonists can produce significant undesirable side effects such as postural hypotension, sedation or depression, increased gastrointestinal motility and diarrhea, impaired ability to ejaculate, nasal stuffiness, akinesia and the like.
Compounds that interact more selectively with a particular alpha1 adrenergic receptor subtype may prove clinically useful in providing more selective treatment of conditions and diseases associated with abnormal activity at the receptor subtype. For example, alpha1 adrenergic receptor antagonists that can selectively ameliorate nociceptive and/or neurogenic pain without affecting blood pressure or causing postural hypotension, are desirable. Presently available alpha1 adrenergic antagonists are either relatively nonselective with respect to the subtypes with which they interact or are not selective for the alpha1B adrenergic receptor subtype.
Selective alpha1B adrenergic receptor antagonists can also be useful in the treatment of CNS disorders including, but not limited to, anxiety, sleep disorders, and schizophrenia. (See, e.g., Bakshi et al. (1999) Neuroscience 92:113-121; Carasso, et al. (1998) Neuropharmacol. 37:401-404; and Acosta-Martinez, et al. (1999) Neurochem. Int. 35:383-391.)
The present invention relates to the discovery of a preferred class of novel compounds that are selective alpha1B adrenergic receptor antagonists (see, commonly owned U.S. patent applications U.S. S.No. 60/124,781; filed on Mar. 17, 1999; U.S. S.No. 60/165,312, filed on Nov. 12, 1999; and U.S. Ser. No. 09/521,185, filed Mar. 8, 2000 by Coffen, et al. entitled xe2x80x9cOxazolone Derivatives and Uses Thereof,xe2x80x9d filed herewith, the disclosure of which are incorporated by reference herein), and that alpha1B adrenergic receptor-selective compounds possess analgesic activity.
Accordingly, there is a need in the art for a method by which compounds can be identified having alpha1B adrenergic receptor-mediated analgesic activity.
It is a primary object of the invention to addressed the above-described need in the art.
It is another object of the invention to provide a method by which compounds can be screened to identify those that produce alpha1B adrenergic receptor-mediated analgesia.
It is still another object of the invention to provide compounds identified by the aforementioned method.
It is yet another object of the invention to provide a method of treating a subject to produce analgesia comprising administering to the subject in need of analgesia a therapeutically effective amount of a compound identified by the aforementioned method, or a pharmaceutically acceptable salt or hydrate thereof, or pharmaceutical composition comprising such a compound, or salt or hydrate thereof.
In one embodiment of the invention, a method for screening for compounds having alpha1B adrenergic receptor-mediated analgesic activity is provided. The method comprises (a) measuring the activity of a test compound in a first binding assay and (b) measuring activity of the test compound in at least one pain model, wherein (a) and (b) are done concurrently or consecutively in any order. The first binding assay comprises (i) providing a preparation of a cell that expresses an alpha1B adrenergic receptor, (ii) combining a test compound with the cell preparation, and (iii) measuring binding of the test compound to the cell preparation or the receptor. The test compound is also be evaluated in a second binding assay for its ability to bind to the alpha1A and/or alpha1D adrenergic receptor, and/or to other receptors, e.g., histamine receptors, dopamine receptors, and the like. A test compound is also evaluated for its ability to elicit an appropriate response in at least one pain model, e.g., the ability to reduce nociceptive or neurogenic pain. Optionally, test compounds are evaluated for other alpha1 adrenergic receptor-mediated functional activity.
The invention includes a method of treating a subject for the production of analgesia. The phrases xe2x80x9cproduction of analgesiaxe2x80x9d and xe2x80x9creduction of painxe2x80x9d are used interchangeably herein. In the method, a therapeutically effective amount of a compound identified by the method disclosed and claimed herein, or a pharmaceutically acceptable salt or hydrate thereof, is administered to a subject in need of a reduction of pain. The compound can be administered after a trauma that causes acute or chronic pain. Optionally, the compound, or a pharmaceutically acceptable salt or hydrate thereof, can be administered as a composition comprising one or more pharmaceutically acceptable additives, diluents, or carriers to form a pharmaceutical composition. In one embodiment, such a composition is a sustained release formulation.
In a further embodiment, the pharmaceutical composition can also include one or more compounds having at least one of antiinflammatory activity, analgesic activity, and anticonvulsant activity. Examples of other compounds include, but are not limited to the following: compounds used in the treatment of neuropathic pain including, but not limited to tricyclic antidepressants (e.g., amitriptyline, imipramine, desipramine), anti-convulsants (e.g., gabapentin, carbamazepine, phenytoin) and local anesthetics (e.g., mexiletine, lidocaine); and compounds used in the treatment of inflammatory pain including, but not limited to nonsteroidal antiinflammatory agents, (e.g., ibuprofen, naprosyn sodium, aspirin, diclofenac sodium, indomethacin, toletin), steroids (e.g., methylprednisone, prednisone), analgesics (e.g., acetaminophen), and opiates (e.g., tramadol, demerol, darvon, vicodin, fentanyl).
Administration of a selected compound is accomplished by conventional means including but not limited to oral, enteral, rectal, mucosal, percutaneous, and parenteral administration. In a preferred embodiment, the selected compound is administered orally.
In another aspect, the invention includes a pharmaceutical composition, comprising (a) a therapeutically effective amount of a compound identified by the method disclosed and claimed herein, or a pharmaceutically acceptable salt or hydrate thereof, and (b) a pharmaceutically acceptable additive, diluent, or carrier. In one embodiment, this composition is a sustained release formulation.
In another embodiment, the present invention encompasses a method of treating a subject suffering from a CNS disorder comprising administering to the subject a therapeutically effective amount of a compound identified by the method of screening disclosed or a pharmaceutically acceptable salt or hydrate thereof. In a further embodiment, the subject suffering from a CNS disorder is administered a therapeutically effective amount of a composition comprising a compound identified by the method of screening disclosed or a pharmaceutically acceptable salt or hydrate thereof and a pharmaceutically acceptable carrier.
The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, I9th edition, Easton, Pennsylvania; Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); Textbook of Pain (P. D. Wall and R. Meizack, editors, Third Edition, Churchill Livingstone, 1994); and Winzor et al. (1995) Quantitative Characterization of Ligand Binding (Wiley-Liss, Inc., NY).
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
As used in this specification and the appended claims, the singular forms xe2x80x9ca,xe2x80x9d xe2x80x9canxe2x80x9d and xe2x80x9cthexe2x80x9d include singular and plural references unless the content clearly dictates otherwise. Thus, for example, reference to xe2x80x9can antagonistxe2x80x9d may include a mixture of two or more such agents.
I. Definitions
In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.
A xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d means a carrier that is useful in preparing a pharmaceutical composition that is generally compatible with the other ingredients of the composition, not deleterious to the recipient, and neither biologically nor otherwise undesirable, and includes a carrier that is acceptable for veterinary use as well as human pharmaceutical use. A xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d as used in the specification and claims includes both one and more than one such carrier.
A xe2x80x9cpharmaceutically acceptable saltxe2x80x9d of a compound means a salt that is pharmaceutically acceptable, as described above, and that possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, benzenesulfonic acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, camphorsulfonic acid, p-chlorobenzenesulfonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hexanoic acid, heptanoic acid, (o-hydroxybenzoyl) benzoic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, lauryl sulfuric acid, malic acid, maleic acid, malonic acid, mandelic acid, methanesulfonic acid, 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, 4,4xe2x80x2-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), muconic acid, 2-napthalenesulfonic acid, oxalic acid, 3-phenylpropionic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, trimethylacetic acid, tertiary butylacetic acid, p-toluenesulfonic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. The preferred pharmaceutically acceptable salts are the salts formed from hydrochloric acid, sulphuric acid, methanesulfonic acid, maleic acid, phosphoric acid, tartaric acid, citric acid, sodium, potassium, calcium, zinc, and magnesium.
Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, and sodium hydroxide. Acceptable organic bases include diethanolamine, ethanolamine, N-methyl-glucamine, triethanolamine, tromethamine, and the like.
xe2x80x9cPharmaceutically acceptable hydratesxe2x80x9d means hydrates, which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such hydrates are formed by the combination of one or more molecules of water with one of the substances, in which the water retains its molecular state as H2O, such combination being able to form one or more than one hydrate.
A xe2x80x9ctherapeutically effective amountxe2x80x9d means an amount of a compound that, when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease. The xe2x80x9ctherapeutically effective amountxe2x80x9d will vary depending on the compound, and disease state being treated, the severity of the disease treated, the age and relative health of the subject, the route and form of administration, the judgement of the attending medical or veterinary practitioner, and other factors.
xe2x80x9calpha1 adrenergic receptorsxe2x80x9d, xe2x80x9calpha1A adrenergic receptorsxe2x80x9d (previously known as xe2x80x9calpha1C adrenergic receptorsxe2x80x9d), xe2x80x9calpha1B adrenergic receptors,xe2x80x9d xe2x80x9calpha1D adrenergic receptorsxe2x80x9d, or xe2x80x9calpha1L adrenergic receptorsxe2x80x9d used interchangeably with xe2x80x9calpha1 adrenoceptorsxe2x80x9d, xe2x80x9calpha1A adrenoceptorsxe2x80x9d (xe2x80x9calpha1C adrenoceptorsxe2x80x9d), xe2x80x9calpha1B adrenoceptorsxe2x80x9d, xe2x80x9calpha 1D adrenoceptorsxe2x80x9d, and xe2x80x9calpha1L adrenoceptorsxe2x80x9d, respectively, refers to a molecule conforming to the seven membrane-spanning G-protein receptors, which under physiologic conditions mediate various actions, for example, of the central and/or peripheral sympathetic nervous system through the binding of the catecholamines, epinephrine and norepinephrine. Examples of physiological effects mediated by alpha1 adrenoceptors include, but are not limited to, control of blood pressure, glycogenolysis, growth and hypertrophy of cardiac myocytes, contractility of the urinary tract, and the like.
The term xe2x80x9calpha1 adrenergic receptor subtypexe2x80x9d used interchangeably with xe2x80x9calpha1 adrenoceptor subtypexe2x80x9d refers to a distinct member of the class of alpha1 adrenoceptors, selected from the alpha1A (previously known as alpha1C), alpha1B, alpha1D and alpha1L adrenoceptors. The subtypes have been distinguished based on differential binding profiles of ligands, such as the agonist oxymetazoline, and such as the antagonists, WB4101 and phentolamine. Furthermore, the genes encoding the alpha1A (previously known as alpha1C), alpha1B and alpha1D subtypes have been isolated and cloned. The existence of an additional subtype, the alpha1L adrenergic receptor subtype, has been proposed, however, the gene for the alpha1L adrenergic receptor subtype has not yet been cloned.
The term xe2x80x9cspecific alpha1 adrenergic receptorxe2x80x9d as used herein, refers to a distinct member of the group or class of adrenoceptors, which may be selected from the human alpha1A (previously known as alpha1C), alpha1B, alpha1D and alpha1L adrenoceptors. Preferred species from which may be derived or isolated alpha1 adrenergic receptor subtype polypeptides, genes encoding an alpha1 adrenergic receptor subtype, and/or cells, tissues and organs that express one or more alpha1 adrenergic receptor subtype, include human, bovine, rat, murine, porcine, ovine, and the like. A more preferred species is human.
xe2x80x9cAlpha1B adrenergic receptorsxe2x80x9d means a specific alpha1 adrenoceptor expressed in numerous tissues, most notably in the liver, heart, cerebral cortex, and spinal cord.
The term xe2x80x9ccell preparationxe2x80x9d as used herein with respect to the expression of an alpha1 adrenergic receptor or an alpha1 adrenergic receptor subtype intends any form of a cell that expresses the receptor or receptor subtype of interest in which receptor- or receptor subtype-specific binding of a ligand, a radioligand, a test compound, or the like, can be measured. The term xe2x80x9ccell preparationxe2x80x9d includes, but is not limited to, a tissue explant, an intact cell isolated from a subject or tissue, an intact cultured or recombinant cell, as well as a disrupted cell, e.g., a cell membrane homogenate, a solubilized cell membrane in which receptor-specific binding can be measured, and the like.
The term xe2x80x9cpharmacological effectxe2x80x9d as used herein encompasses effects produced in the subject that achieve the intended purpose of a therapy. In preferred embodiments, a pharmacological effect means that pain symptoms of the subject being treated are prevented, alleviated, or reduced. For example, a pharmacological effect would be one that results in the production of analgesia in a treated subject.
xe2x80x9cTreatingxe2x80x9d or xe2x80x9ctreatmentxe2x80x9d of a disease includes: (1) preventing the disease, i.e. causing the clinical symptoms of the disease not to develop in a subject that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease, (2) inhibiting the disease, i.e., arresting the development of the disease or its clinical symptoms, or (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms.
The term xe2x80x9csubjectxe2x80x9d as used herein encompasses mammals and non-mammals. Examples of mammals include, but are not limited to: any member of the Mammalia class, including humans, nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs, and the like. Examples of nonmammals include, but are not limited to, birds, reptiles, and the like. The term does not denote a particular age or sex.
xe2x80x9cTraumaxe2x80x9d means any wound or injury. Trauma can produce, for example, acute and/or chronic pain, inflammatory pain, and neuropathic pain.
xe2x80x9cPainxe2x80x9d means more or less localized sensation of discomfort, distress, or agony, resulting from the stimulation of specialized nerve endings. There are many types of pain, including, but not limited to, lightning pains, phantom pains, shooting pains, acute pain, inflammatory pain, neuropathic pain, complex regional pain, neuralgia, neuropathy, and the like (Dorland""s Illustrated Medical Dictionary, 28th Edition, W. B. Saunders Company, Philadelphia, Pa.). The goal of treatment of pain is to reduce the degree or severity of pain perceived by a treatment subject.
xe2x80x9cNeuropathic painxe2x80x9d means the pain resulting from functional disturbances and/or pathological changes as well as noninflammatory lesions in the peripheral nervous system. Examples of neuropathic pain include, but are not limited to, thermal or mechanical hyperalgesia, thermal or mechanical allodynia, diabetic pain, entrapment pain, and the like.
xe2x80x9cHyperalgesiaxe2x80x9d means the abnormally increased pain sense, such as pain that results from an excessive sensitiveness or sensitivity.
xe2x80x9cHypalgesiaxe2x80x9d (or xe2x80x9chypoalgesiaxe2x80x9d) means the decreased pain sense.
xe2x80x9cAllodyniaxe2x80x9d means the pain that results from a non-noxious stimulus to the skin. Examples of allodynia include, but are not limited to, cold allodynia, tactile allodynia, and the like.
xe2x80x9cComplex regional pain syndromesxe2x80x9d means the pain that includes, but is not limited to, reflex sympathetic dystrophy, causalgia, sympathetically maintained pain, and the like.
xe2x80x9cCausalgiaxe2x80x9d means the burning pain, often accompanied by trophic skin changes, due to injury of a peripheral nerve.
xe2x80x9cNociceptionxe2x80x9d is defined herein as pain sense. xe2x80x9cNociceptorxe2x80x9d herein refers to a structure that mediates nociception. The nociception may be the result of a physical stimulus, such as, mechanical, electrical, thermal, or a chemical stimulus. Most nociceptors are in either the skin or the viscera walls.
xe2x80x9cAnalgesiaxe2x80x9d is defined herein as the relief of pain without the loss of consciousness. An xe2x80x9canalgesicxe2x80x9d is an agent or drug useful for relieving pain, again, without the loss of consciousness.
xe2x80x9cIsomerismxe2x80x9d means the compounds have identical molecular formulae but that differ in the nature or sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed xe2x80x9cstereoisomersxe2x80x9d. Stereoisomers that are not mirror images of one another are termed xe2x80x9cdiastereomersxe2x80x9d and stereoisomers that are nonsuperimposable mirror images are termed xe2x80x9cenantiomersxe2x80x9d or sometimes optical isomers. A carbon atom bonded to four nonidentical substituents is termed a xe2x80x9cchiral center.xe2x80x9d
xe2x80x9cChiral isomerxe2x80x9d means a compound with one chiral center. It has two enantiomeric forms of opposite chirality, and may exist as either an individual enantiomer or as a mixture of enantiomers. A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is termed a xe2x80x9cracemic mixture.xe2x80x9d A compound that has more than one chiral center has 2nxe2x88x921 enantiomeric pairs, where n is the number of chiral centers. Compounds with more than one chiral center may exist as either an individual diastereomer or as a mixture of diastereomers, termed a xe2x80x9cdiastereomeric mixture.xe2x80x9d
When one chiral center is present a stereoisomer may be characterized by the absolute configuration (R) or (S) of that chiral center. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn et al. Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al. Angew. Chem. 1966, 78, 413; Cahn and Ingold J. Chem. Soc. (London) 1951, 612; Cahn et al. Experientia 1956, 12, 81; Cahn, J. Chem. Educ. 1964, 41, 116).
xe2x80x9cGeometric isomersxe2x80x9d means the diastereomers that owe their existence to hindered rotation about double bonds. Their configurations are differentiated in their names by prefixes cis and trans, or Z and E, which indicate that the groups are on the same or opposite side of the double bond in the molecule according to the Cahn-Ingold-Prelog rules.
xe2x80x9cAtropic isomersxe2x80x9d means the isomers owing their existence to restricted rotation caused by hindrance of rotation of large groups about a central bond.
Reference to a xe2x80x9ccompoundxe2x80x9d includes individual isomers as well as racemic or nonracemic mixtures of isomers, and pharmaceutically acceptable salts or hydrates thereof.
xe2x80x9cOptionalxe2x80x9d or xe2x80x9coptionallyxe2x80x9d means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, the phrase xe2x80x9coptionally, test compounds are evaluated for other alpha1 adrenergic receptor-mediated functional activityxe2x80x9d means that the compound referred to may or may not be evaluated as described in order to fall within the scope of the invention, and that the description includes the situation wherein the compound is evaluated and the situation in which the compound is not evaluated for other alpha1 adrenergic receptor mediated functional activity.
Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations, process parameters, or the order in which method steps are performed as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.
Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.
A. Pharmacology and Utility
The present invention is based on the unexpected finding that selective alpha1B adrenergic receptor antagonists have specific analgesic activity.
1. Selective Alpha1B Adrenergic Receptor Binding Activity
The receptor specificity of a test compound can be determined by any method well known in the art. In one preferred embodiment, the binding of compounds at different receptors, including but not limited to, human, bovine, rat, murine, porcine, ovine, and the like, receptors, preferably human receptors, can be determined in vitro using cultured cell lines that selectively express the receptor of interest.
Cell Lines That Express Receptors of Interest
Examples of cell lines that are available from the American Type Culture Collection (ATCC) that express human receptors as follows (ATCC Accession No.): alpha1A (CRL 11138), alpha1B (CRL 11139), alpha1C (CRL 11140), alpha 2A (CRL 11180), alpha2B (CRL 10275, CRL 11888), alpha2C (CRL 11181), 5-HT1D1 (CRL 10421), 5-HT1D2 (CRL 10422), 5-HT1E (CRL 10913), 5-HT 1F (CRL 10957), 5-HT4B (CRL 11166), 5-HT1A (CRL 11889) and 5-HT2 (CRL 10287).
In addition, plasmids are available from the ATCC that harbor cloned human D2, H2 and H1 receptors (ATCC Accession Nos. 7534, 75345 and 75346, respectively) that can be used to transfect cells using conventional methods well known in the art to provide cells that express on their surface the receptor of interest. Transient transfection of COS-7 cells with a plasmid can be performed using any method known in the art, e.g., the DEAE-dextran method. Briefly, a plasmid comprising an expression vector for the receptor of intrest is added to monolayers of COS-7 cells bathed in DEAE-dextran solution. To enhance the efficiency of transfection diemthyl sulfoxide can be added (Lopata et al. (1984) Nucleic Acids Res. 12:5707-5717). Cells are then grown under controlled conditions and used in experiments after about 72 hours. Stable cells lines can also be obtained by methods well known in the art. For example, a suitable host cell can be cotransfected using, e.g., the calcium phosphate method, with a plasmid comprising an expression vector for the receptor of interest and a plasmid comprising a gene that allows for selection of successfully tranfected cells. Cells are then grown in a controlled environment and selected for expression of the receptor of interest. By continuing to grow and select cells stable, cell lines can be obtained expressing the receptor of interest.
Additional reference with respect to the preparation of DNA encoding these receptors and recombinant cells expressing the DNA can be made to U.S. Pat. No. 5,610,174 to Craig et al., U.S. Pat. Nos. 5,556,753 and 5,714,381 to Bard et al., U.S. Pat. Nos. 5,155,218, 5,360,735, 5,476,782, 5,595,880, 5,639,652, 5,652,113, 5,786,155 and 5,786,157 to Weinshank et al., and U.S. Pat. Nos. 5,403,847, 5,578,611, and 5,780,485 to Gluchowski et al.
Binding Assays
The binding of a test compound to a receptor of interest can be detected by use of a radioligand selective for the receptor. Any radioligand binding technique known in the art may be used to detect the receptor (see, e.g., Winzor et al. (1995) Quantitative Characterization of Ligand Binding, Wiley-Liss, Inc., NY). Binding assays, the evaluation of analgesic activity of a test compound, and/or the evaluation of other functional alpha1 adrenergic receptor-mediated activity of a test compound, as described hereinbelow, can be carried out concurrently and/or consecutively, in any order.
The binding of a test compound to a receptor of interest can be evaluated by, e.g., competitive binding assays using membrane preparations derived from cells that express the receptor or, alternatively, intact cells that express the receptor can be used. First, conditions are determined that allow measurement of the specific binding of a compound known to bind to the receptor. Then, the binding of the known compound to the receptor in a membrane preparation or intact cell preparation is evaluated in the presence of several different concentrations of the test compound. Binding of the test compound to the receptor results in a diminution in the amount of the known compound that binds to the receptor. A test compound having a high affinity for the receptor of interest displaces a given fraction of the bound known compound at a concentration lower than the concentration that would be required if the test compound has a low affinity for the receptor of interest. Thus, for example, competitive radioligand binding assays can be done using the following radioligand/compound combinations for determining specific and nonspecific binding for receptors of interest: [3H]prazosin/phentolamine (alpha1); [3H]rauwolscine/phentolamine (alpha2); [3H]mepyramine/mepyramine (H1); [3H]tiotidine/histamine (H2); [3H]serotonin/serotonin (5-HT1); [3H]ketanserin/mianserin (5-HT2);[3H]-8-OH-DPAT/mianserin (5-H1A); and [3H]spiperone/(+)butaclomol (D2).
2. Pain Models
The analgesic activity of a test compound can be evaluated by any method known in the art. Such methods are described in detail in Examples 1 through 12, below. The evaluation of analgesic activity of a test compound, and/or the evaluation of other functional alpha1 adrenergic receptor-mediated activity of a test compound, as described hereinbelow, and the binding activity of a test compound, as described above, can be carried out concurrently and/or consecutively, in any order.
3. Motor Activity
The effects of alpha1B adrenergic receptor antagonists on general locomotor activity and sedation can be examined. Locomotor deficits or sedation are both common side effects of potential analgesic compounds. The locomotor activity test is described in Example 13, below.
4. Functional Activity
The functional alpha1-adrenergic receptor activity of a test compound can be determined by art-recognized procedures. Such studies are conducted to determine whether a test compound exhibits agonist or antagonist activity at alpha1 adrenergic receptor. Such activity can be indicative of potential side effects. Examples of in vitro and in vivo assays for measuring the relative effect of a test compound on alpha1 adrenergic receptor-mediated activity, either agonist or antagonist activity, are described in Examples 14 through 18, below.
5. Neuropsychiatric Disorders
The assay of the present invention can be employed to screen for compounds useful in the treatment of neuropsychiatric disorders. These disorders include, but are not limited to, generalized anxiety, sleep disorders, and schizophrenia. Animal models are also available to concurrently or subsequently test potential therapeutics found by the assay of the present invention. These animal models are well known in the art and are described, e.g., in Bakshi, et al. (1999) supra; Carasso, et al. (1998) supra; and Acosta-Martinez, et al. (1999) supra.
B. Administration and Pharmaceutical Compositions
A compound identified as a selective alpha1B adrenergic receptor antagonist by the method disclosed and claimed herein, when employed in the prevention or treatment of pain or neuropsychiatric disorders, can be formulated neat or with the addition of pharmaceutically acceptable excipients, additives, diluents, or carriers (see, for example, formulations as described in Remington: The Science and Practice of Pharmacy, supra.
The invention includes a pharmaceutical composition comprising at least one compound identified as a selective alpha1B adrenergic receptor antagonist by the method disclosed and claimed herein or a pharmaceutically acceptable salt, hydrate, or derivative thereof together with one or more pharmaceutically acceptable carriers, and optionally other therapeutic and/or prophylactic ingredients.
In general, a compound identified as a selective alpha1B adrenergic receptor antagonist by the method disclosed and claimed herein will be administered in a therapeutically effective amount by any of the accepted modes of administration for agents that serve similar utilities. Suitable dosage ranges are about 1-500 mg daily, preferably about 1-100 mg daily, and more preferably about 1-30 mg daily, depending upon numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound used, the route and form of administration, the indication towards which the administration is directed, and the preferences and experience of the medical or veterinary practitioner involved. One of ordinary skill in the art of treating such diseases will be able, without undue experimentation and in reliance upon personal knowledge and the disclosure of this application, to ascertain a therapeutically effective amount of the compounds of this invention for a given disease.
In general, a compound identified as a selective alpha1B adrenergic receptor antagonist by the method disclosed and claimed herein will be administered as pharmaceutical formulations including those suitable for oral (including buccal and sub-lingual), rectal, nasal, topical, pulmonary, vaginal or parenteral (including intramuscular, intraarterial, intrathecal, subcutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation. The preferred manner of administration is oral using a convenient daily dosage regimen which can be adjusted according to the degree of affliction.
A compound identified as a selective alpha1B adrenergic receptor antagonist by the method disclosed and claimed herein, together with a conventional adjuvant, carrier, or diluent, may be placed into the form of pharmaceutical compositions and unit dosages. The pharmaceutical compositions and unit dosage forms may comprise of conventional ingredients in conventional proportions, with or without additional active compounds or principles, and the unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed. The pharmaceutical composition may be employed as solids, such as tablets or filled capsules, semisolids, powders, sustained release formulations, or liquids such as solutions, suspensions, emulsions, elixirs, or filled capsules for oral use; or in the form of suppositories for rectal or vaginal administration; or in the form of sterile injectable solutions for parenteral use. Formulations containing about 0.01 to 100 milligrams, preferably about 1 to 50 milligrams, more preferably about 1 to 10 milligrams, per tablet, are accordingly suitable representative unit dosage forms.
A compound identified as a selective alpha1B adrenergic receptor antagonist by the method disclosed and claimed herein may be formulated in a wide variety of oral administration dosage forms. The pharmaceutical compositions and dosage forms may comprise the compound or its pharmaceutically acceptable salt or hydrate as the active component. The pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. In powders, the carrier is a finely divided solid which is a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably containing from one to about seventy percent of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term xe2x80x9cpreparationxe2x80x9d is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be as solid forms suitable for oral administration.
Other forms suitable for oral administration include liquid form preparations including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, or solid form preparations which are intended to be converted shortly before use to liquid form preparations. Emulsions may be prepared in solutions in aqueous propylene glycol solutions or may contain emulsifying agents such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents. Aqueous suspensions can be prepared by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well known suspending agents. Solid form preparations include solutions, suspensions, and emulsions, and may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
A compound identified as a selective alpha1B adrenergic receptor antagonist by the method disclosed and claimed herein may be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Examples of oily or nonaqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and may contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.
A compound identified as a selective alpha1B adrenergic receptor antagonist by the method disclosed and claimed herein may be formulated for topical administration to the epidermis as ointments, creams or lotions, or as a transdermal patch. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also containing one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. Formulations suitable for topical administration in the mouth include lozenges comprising active agents in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
A compound identified as a selective alpha1B adrenergic receptor antagonist by the method disclosed and claimed herein may be formulated for administration as suppositories. A low melting wax, such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active component is dispersed homogeneously, for example, by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and to solidify.
A compound identified as a selective alpha1B adrenergic receptor antagonist by the method disclosed and claimed herein may be formulated for vaginal administration. Pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
A compound identified as a selective alpha1B adrenergic receptor antagonist by the method disclosed and claimed herein may be formulated for nasal administration. The solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The formulations may be provided in a single or multidose form. In the latter case of a dropper or pipette this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray this may be achieved for example by means of a metering atomizing spray pump.
A compound identified as a selective alpha1B adrenergic receptor antagonist by the method disclosed and claimed herein may be formulated for aerosol administration, particularly to the respiratory tract and including intranasal administration. The compound will generally have a small particle size for example of the order of about 5 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization. The active ingredient is provided in a pressurized pack with a suitable propellant such as a chlorofluorocarbon (CFC) for example dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoro-ethane, carbon dioxide or other suitable gas. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of compound may be controlled by a metered valve. Alternatively the active ingredients may be provided in a form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP). The powder carrier will form a gel in the nasal cavity. The powder composition may be presented in unit dose form for example in capsules or cartridges of e.g., gelatin or blister packs from which the powder may be administered by means of an inhaler.
When desired, formulations can be prepared with enteric coatings adapted for sustained or controlled release administration of the active ingredient.
The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
Other suitable pharmaceutical carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, supra. Representative pharmaceutical formulations containing a compound of the present invention are described in Example 19.
In addition to being administered alone, an alpha1B adrenergic receptor antagonist can be administered for the treatment of pain in combination with other appropriate compounds, such as, analgesics or anticonvulsants. Such compounds include, but are not limited to the following: agents used in the treatment of neuropathic pain including, but not limited to, tricyclic antidepressants (e.g., amitriptyline, imipramine, desipramine), anticonvulsants (e.g., gabapentin, carbamazepine, phenytoin) and local anesthetics (e.g., mexiletine, lidocaine); and agents used in the treatment of inflammatory pain including, but not limited to, NSAIDs (e.g., ibuprofen, naprosyn sodium, aspirin, diclofenac sodium, indomethacin, toletin), steroids (e.g., methylprednisone, prednisone), analgesics (e.g., acetaminophen), and opiates (e.g., tramadol, demerol, darvon, vicodin, fentanyl).
In view of the guidance provided herein concerning the present invention, other compounds, including other alpha adrenergic receptor ligands, that block alpha1B adrenergic receptors can be examined by the teachings of the present specification to determine their usefulness in the treatment of pain. Determination of a compound""s ability to selectively block alpha1B adrenergic receptors can be performed as described in Example 1. The usefulness of the compound for the treatment of pain can be evaluated as described herein in Examples 2-6. Binding assays, the evaluation of analgesic activity of a test compound, and/or the evaluation of other functional alpha1 adrenergic receptor-mediated activity of a test compound, as described herein, can be carried out concurrently and/or consecutively, in any order, and near or remote in time.
As stated above, the role of alpha1B adrenergic receptor antagonists in analgesia has not been previously described. Experiments performed in support of the present invention suggest that alpha1B adrenergic receptor antagonists may be useful in the treatment of acute, inflammatory, and neuropathic pain in a subject. Furthermore, compounds identified through the assay of the present invention are useful in the treatment of neuropsychiatric disorders.
Below are examples of specific embodiments of the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.